水稻种子携带稻瘟病菌LAMP检测方法的建立与应用

Development and application of loop-mediated isothermal amplification method for detection of Magnaporthe oryzae from rice seeds

稻瘟病菌Magnaporthe oryzae可通过种子带菌实现远距离传播,导致病害发生并造成严重经济损失。建立灵敏高效且便于操作的水稻种子携带稻瘟病菌的检测方法,是确保种子健康和控制病害发生的先决条件。本研究选取稻瘟病菌的3-磷酸甘油酰基转移酶基因MoSPC3设计合成3对特异性引物,并对反应体系进行优化,建立了环介导等温扩增(loop-mediated isothermal amplification,LAMP)方法。该方法可特异性地检测稻瘟病菌,对稻瘟病菌DNA样品的检测灵敏度可达到0.24拷贝/μL,远高于普通PCR的检测灵敏度。将该方法用于从中国和马来西亚不同水稻种植区收集的92份水稻种子样品携带稻瘟病菌的检测,结果显示,LAMP方法共检测到89份阳性样品,而普通PCR仅显示有2份样品为阳性。综上,本研究建立了一种适用于水稻种子携带稻瘟病菌的LAMP检测方法,具有简便快捷、灵敏度高等特点,丰富了稻瘟病菌的检测体系,可用于大批量水稻种子样品的健康检测。

The fungus Magnaporthe oryzae,as the causal agent of rice blast disease,can be transmitted by rice seeds for a long distance and caused serious economic losses in rice production.To establish a sensitive,effective and convenient method for detection of M.oryzae from seeds is a precondition for rice seed health and control of rice blast disease.In this study,three primer pairs were designed based on glycerol-3-phosphate acyltransferase gene(MoSPC3)sequence of M.oryzae,and the loop-mediated isothermal amplification(LAMP)reaction system was developed.The results showed that the LAMP method was specific for detection of M.oryzae,and the sensitivity was about 0.24 copies/μL DNA,which was much more sensitive than PCR method.A total of 92 rice seed samples collected from different production areas of China and Malaysia were examined by the LAMP detection system.The results showed that 89 samples were M.oryzae positive by LAMP detection,while only two samples were detected positive by ordinary PCR.In conclusion,a LAMP system for detection of M.oryzae from rice seeds was developed,which was more convenient and more sensitive than ordinary PCR.It broadened the detection strategies for M.oryzae and could be applied for large-scale seeds health test.